The influence of extracellular calcium concentrations ([Ca(2+)]) on single platelet activation and platelet aggregation was evaluated. Platelet fibrinogen binding and P-selectin expression were monitored by whole blood flow cytometry in the presence of EDTA or 0.125, 1.25, 2.5, 5, or 10 mM [Ca(2+)] and in the absence or presence of the thromboxane A(2) (TXA(2)) blockade. Platelet aggregation was measured in citrated and hirudinized platelet-rich plasma (PRP). Platelet fibrinogen binding was slightly increased at >/=2.5 mM [Ca(2+)] in unstimulated samples. ADP-induced platelet fibrinogen binding was, however, higher at 0.125 mM, but lower at 5 and 10 mM [Ca(2+)], as compared to 1.25 mM [Ca(2+)]. Platelet P-selectin expression was not affected by extracellular [Ca(2+)], except mild increases of ADP-induced platelet P-selectin expression in the presence of EDTA. TXA(2) blockade by ICI 192.605 influenced above flow cytometric analyses little. Using Born aggregometry, ADP induced more intense platelet aggregation in citrated PRP than in hirudinized PRP. TXA(2) blockade did not affect platelet aggregation in hirudinized PRP, but reduced aggregation in citrated PRP to approximately 85% of that in hirudinized samples. ADP also induced a more marked and sustained elevation of intracellar [Ca(2+)] in the presence of extracellular [Ca(2+)]. Thus, extracellular [Ca(2+)] has little influence on flow cytometric analysis of platelet P-selectin expression. High [Ca(2+)] enhances spontaneous platelet fibrinogen binding, but reduces ADP-induced platelet fibrinogen binding, while low [Ca(2+)] enhances ADP-induced platelet fibrinogen binding. Physiological [Ca(2+)] supports more intense platelet aggregation when effect of artificial TXA(2) synthesis is blocked.